Mitomycin C (MC) is a potent antibiotic with increasing clinical interest as an anticancer agent. The mechanism of its cytotoxic activities is proposed to be related to the monofunctional and bifunctional binding of MC to cellular macromolecules, and MC is stimulation of cellular generation of active oxygen products. The objective of this research project is to ascertain the molecular mechanisms of the activation of MC and the interaction of the activated MC with DNA in vitro and in intact cells. Direct identification of reactive sites of the MC molecules and the covalently linked adducts formed with DNA in vitro and in intact cell will provide information regarding the mechanism of cytotoxicity of MC. In vitro activation of MC by two flavo-enzymes generates a quinone radical intermediate which then is attacked by nucleophiles such as DNA, resulting in monofunctional and bifunctional bindings. One of the reactive sites of MC has been proven to be at the C-1 position of MC while the second reactive site required for the bifunctional binding is still unclear. This site will be studied and defined through the activation of primary MC metabolites. The binding site of DNA is proposed to be 0-6 position of guanine. In vitro formed MC-DNA adducts, including monofunctional and bifunctional adducts generated by flavo-enzyme activated MC will be isolated and identified. Kinetics of the formation of the adducts, and the identity of MC-adducts will provide the information for the elucidation of the mechanism of MC binding to DNA. MC-DNA adduct formation and its removal in intact cells under hypoxic and oxygenated conditions will be evaluated and compared. The results will ascertain the effect of aerobic and anaerobic conditions on cytotoxicity of MC (or porfiromycin).